Induced pluripotent stem cell derived pericytes respond to mediators of proliferation and contractility.

BACKGROUND: Pericytes are multifunctional contractile cells that reside on capillaries. Pericytes are critical regulators of cerebral blood flow and blood-brain barrier function, and pericyte dysfunction may contribute to the pathophysiology of human neurological diseases including Alzheimers disease, multiple sclerosis, and stroke. Induced pluripotent stem cell (iPSC)-derived pericytes (iPericytes) are a promising tool for vascular research. However, it is unclear how iPericytes functionally compare to primary human brain vascular pericytes (HBVPs). METHODS: We differentiated iPSCs into iPericytes of either the mesoderm or neural crest lineage using established protocols. We compared iPericyte and HBVP morphologies, quantified gene expression by qPCR and bulk RNA sequencing, and visualised pericyte protein markers by immunocytochemistry. To determine whether the gene expression of neural crest iPericytes, mesoderm iPericytes or HBVPs correlated with their functional characteristics in vitro, we quantified EdU incorporation following exposure to the key pericyte mitogen, platelet derived growth factor (PDGF)-BB and, contraction and relaxation in response to the vasoconstrictor endothelin-1 or vasodilator adenosine, respectively. RESULTS: iPericytes were morphologically similar to HBVPs and expressed canonical pericyte markers. However, iPericytes had 1864 differentially expressed genes compared to HBVPs, while there were 797 genes differentially expressed between neural crest and mesoderm iPericytes. Consistent with the ability of HBVPs to respond to PDGF-BB signalling, PDGF-BB enhanced and a PDGF receptor-beta inhibitor impaired iPericyte proliferation. Administration of endothelin-1 led to iPericyte contraction and adenosine led to iPericyte relaxation, of a magnitude similar to the response evoked in HBVPs. We determined that neural crest iPericytes were less susceptible to PDGFR beta inhibition, but responded most robustly to vasoconstrictive mediators. CONCLUSIONS: iPericytes express pericyte-associated genes and proteins and, exhibit an appropriate physiological response upon exposure to a key endogenous mitogen or vasoactive mediators. Therefore, the generation of functional iPericytes would be suitable for use in future investigations exploring pericyte function or dysfunction in neurological diseases.

Brain pericytes in culture display diverse morphological and functional phenotypes.

Pericytes play several important functions in the neurovascular unit including contractile control of capillaries, maintenance of the BBB, regulation of angiogenesis, and neuroinflammation. There exists a continuum of pericyte subtypes along the vascular tree which exhibit both morphological and transcriptomic differences. While different functions have been associated with the pericyte subtypes in vivo, numerous recent publications have used a primary human brain vascular pericytes (HBVP) cell line where this pericyte heterogeneity has not been considered. Here, we used primary HBVP cultures, high-definition imaging, cell motility tracking, and immunocytochemistry to characterise morphology, protein expression, and contractile behaviour to determine whether heterogeneity of pericytes also exists in cultures. We identified five distinct morphological subtypes that were defined using both qualitative criteria and quantitative shape analysis. The proportion of each subtype present within the culture changed as passage number increased, but pericytes did not change morphological subtype over short time periods. The rate and extent of cellular and membrane motility differed across the subtypes. Immunocytochemistry revealed differential expression of alpha-smooth muscle actin (αSMA) across subtypes. αSMA is essential for cell contractility, and consequently, only subtypes with high αSMA expression contracted in response to physiological vasoconstrictors endothelin-1 (ET1) and noradrenaline (NA). We conclude that there are distinct morphological subtypes in HBVP culture, which display different behaviours. This has significance for the use of HBVP when modelling pericyte physiology in vitro where relevance to in vivo pericyte subtypes along the vascular tree must be considered.

Microglia directly associate with pericytes in the central nervous system.

Cerebral blood flow (CBF) is important for the maintenance of brain function and its dysregulation has been implicated in Alzheimer's disease (AD). Microglia associations with capillaries suggest they may play a role in the regulation of CBF or the blood-brain-barrier (BBB). We explored the relationship between microglia and pericytes, a vessel-resident cell type that has a major role in the control of CBF and maintenance of the BBB, discovering a spatially distinct subset of microglia that closely associate with pericytes. We termed these pericyte-associated microglia (PEM). PEM are present throughout the brain and spinal cord in NG2DsRed × CX3 CR1+/GFP mice, and in the human frontal cortex. Using in vivo two-photon microscopy, we found microglia residing adjacent to pericytes at all levels of the capillary tree and found they can maintain their position for at least 28 days. PEM can associate with pericytes lacking astroglial endfeet coverage and capillary vessel width is increased beneath pericytes with or without an associated PEM, but capillary width decreases if a pericyte loses a PEM. Deletion of the microglia fractalkine receptor (CX3 CR1) did not disrupt the association between pericytes and PEM. Finally, we found the proportion of microglia that are PEM declines in the superior frontal gyrus in AD. In summary, we identify microglia that specifically associate with pericytes and find these are reduced in number in AD, which may be a novel mechanism contributing to vascular dysfunction in neurodegenerative diseases.

Pharmacological PDGFRß inhibitors imatinib and sunitinib cause human brain pericyte death in vitro.

Capillary pericytes have numerous functions important for tissue maintenance. Changes in pericyte function are implicated in diseases such as cancer, where pericyte-mediated angiogenesis contributes to the blood supply that tumors use to survive. Some anti-cancer agents, like imatinib, target platelet-derived growth factor receptor-beta (PDGFRß). Healthy pericytes rely on PDGFRß phosphorylation for their survival. Therefore, we hypothesised that pharmacological agents that block PDGFRß phosphorylation could be used to kill pericytes. We treated human brain vascular pericytes, which express PDGFRß, with three receptor tyrosine kinase inhibitors: imatinib, sunitinib and orantinib. Imatinib and sunitinib, but not orantinib, inhibited PDGFRß phosphorylation in pericytes. Imatinib and sunitinib also reduced viability, prevented proliferation, and induced death, while orantinib only blocked pericyte proliferation. Overall, we found that receptor tyrosine kinase inhibitors that block PDGFRß phosphorylation cause healthy pericytes to die in vitro. While useful in cancer to limit tumor growth, these agents could impair healthy brain pericyte survival and impact brain function.

The rise of pericytes in neurovascular research.

The popularity of pericyte research is increasing, and this was not more evident than at the recent 2019 Brain meeting in Yokohama which featured a large number of presentations focused on brain pericyte research, including the Presidential Symposium. In this article, we will provide a history of brain pericyte research, present the results of our analysis showing a substantial increase in brain pericyte research presented at Brain meetings since 2005, suggest reasons for their increased popularity, and comment on what the future holds for brain pericyte research.

Pericytes and Neurovascular Function in the Healthy and Diseased Brain.

Pericytes are multi-functional cells embedded within the walls of capillaries throughout the body, including the brain. Pericytes were first identified in the 1870s, but little attention was paid to them during the following century. More recently, numerous vascular functions of pericytes have been identified including regulation of cerebral blood flow, maintenance of the blood-brain barrier (BBB), and control of vascular development and angiogenesis. Pericytes can also facilitate neuroinflammatory processes and possess stem cell-like properties. Pericytes form part of the neurovascular unit (NVU), a collection of cells that control interactions between neurons and the cerebral vasculature to meet the energy demands of the brain. Pericyte structure, expression profile, and function in the brain differ depending on their location along the vascular bed. Until recently, it has been difficult to accurately define the sub-types of pericytes, or to specifically target pericytes with pharmaceutical agents, but emerging techniques both in vitro and in vivo will improve investigation of pericytes and allow for the identification of their possible roles in diseases. Pericyte dysfunction is increasingly recognized as a contributor to the progression of vascular diseases such as stroke and neurodegenerative diseases such as Alzheimer's disease. The therapeutic potential of pericytes to repair cerebral blood vessels and promote angiogenesis due to their ability to behave like stem cells has recently been brought to light. Here, we review the history of pericyte research, the present techniques used to study pericytes in the brain, and current research advancements to characterize and therapeutically target pericytes in the future.

Low-density Lipoprotein Receptor-related Proteins in a Novel Mechanism of Axon Guidance and Peripheral Nerve Regeneration.

The low-density lipoprotein receptor-related protein receptors 1 and 2 (LRP1 and LRP2) are emerging as important cell signaling mediators in modulating neuronal growth and repair. We examined whether LRP1 and LRP2 are able to mediate a specific aspect of neuronal growth: axon guidance. We sought to identify LRP1 and LRP2 ligands that could induce axonal chemoattraction, which might have therapeutic potential. Using embryonic sensory neurons (rat dorsal root ganglia) in a growth cone turning assay, we tested a range of LRP1 and LRP2 ligands for the ability to guide growth cone navigation. Three ligands were chemorepulsive: α-2-macroglobulin, tissue plasminogen activator, and metallothionein III. Conversely, only one LRP ligand, metallothionein II, was found to be chemoattractive. Chemoattraction toward a gradient of metallothionein II was calcium-dependent, required the expression of both LRP1 and LRP2, and likely involves further co-receptors such as the tropomyosin-related kinase A (TrkA) receptor. The potential for LRP-mediated chemoattraction to mediate axonal regeneration was examined in vivo in a model of chemical denervation in adult rats. In these in vivo studies, metallothionein II was shown to enhance epidermal nerve fiber regeneration so that it was complete within 7 days compared with 14 days in saline-treated animals. Our data demonstrate that both LRP1 and LRP2 are necessary for metallothionein II-mediated chemotactic signal transduction and that they may form part of a signaling complex. Furthermore, the data suggest that LRP-mediated chemoattraction represents a novel, non-classical signaling system that has therapeutic potential as a disease-modifying agent for the injured peripheral nervous system.